Spectroscopic and Computational Characterization of Substrate-Bound Mouse Cysteine Dioxygenase: Nature of the Ferrous and Ferric Cysteine Adducts and Mechanistic Implications

Abstract

Cysteine dioxygenase (CDO) is a mononuclear non-heme Fe-dependent dioxygenase that catalyzes the initial step of oxidative cysteine catabolism. Its active site consists of an Fe(II) ion ligated by three histidine residues from the protein, an interesting variation on the more common 2-His-1-carboxylate motif found in many other non-heme Fe(II)-dependent enzymes. Multiple structural and kinetic studies of CDO have been carried out recently, resulting in a variety of proposed catalytic mechanisms; however, many open questions remain regarding the structure/ function relationships of this vital enzyme. In this study, resting and substrate-bound forms of CDO in the Fe(II) and Fe(III) states, both of which are proposed to have important roles in this enzyme's catalytic mechanism, were characterized by utilizing various spectroscopic methods. The nature of the substrate/active-site interactions was also explored using the cysteine analog selenocysteine (Sec). Our electronic absorption, magnetic circular dichroism, and resonance Raman data exhibit features characteristic of direct S (or Se) ligation to both the high-spin Fe(II) and Fe(III) active-site ions. The resulting Cys (or Sec)-bound species were modeled and further characterized using density functional theory computations to generate experimentally validated geometric and electronic structure descriptions. Collectively, our results yield a more complete description of several catalytically relevant species and provide support for a reaction mechanism similar to that established for many structurally related 2-His-1-carboxylate Fe(II)-dependent dioxygenases.Cysteine dioxygenase (CDO) is a non-heme Fe(II) dioxygenase that catalyzes the first step of oxidative cysteine catabolism, adding both O atoms from molecular oxygen to L-cysteine (Cys) to form cysteine sulfinic acid (1,2). Cys, one of two sulfur-containing amino acids, is a necessary component in synthesizing new proteins and also serves as a precursor for several biologically important molecules, such as coenzyme A and glutathione (3,4). Additionally, products of the CDO reaction are essential for the biosynthesis of pyruvate, taurine, and hypotaurine. Elevated Cys levels are known to have an excitotoxic effect resulting in neuronal damage and also have been implicated in a variety of neurological disorders, including Parkinson's, Alzheimer's, and motor neuron disease, and autoimmune disorders such as rheumatoid arthritis (5-9). Thus, CDO is a vital enzyme both for providing

A Strongly Bound High-Spin Iron(II) Coordinates Cysteine and Homocysteine in Cysteine Dioxygenase

“…Absorption at 380 nm was seen to decrease with concomitant formation of absorption at 640 nm (ε = 630 M −1 cm −1 ) and a clear isosbestic point at 475 nm ( Figure 4). These data are consistent with the data published by Gardner et al 23 and can be explained as binding of cysteine to the endogenous iron(III) form of CDO. This first-order reaction was fitted globally over all wavelengths, and a rate constant of 2.8 s −1 was extracted, implying a secondorder rate constant for the binding of cysteine to iron(III)CDO of 280 M −1 s −1 .…”

Thiol dioxygenase turnover yields benzothiazole products from 2-mercaptoaniline and O2-dependent oxidation of primary alcohols

“…Despite modest spectroscopic perturbations from the increased polorizability of the Se-atom, both EPR and MCD spectroscopy indicate that the substrate-bound Mm CDO complex with cys and sec exhibit near equivalent coordination geometry and symmetry around the Fe-site [19, 46]. However, rapid mixing of O 2 with the sec -bound enzyme produces no detectable dioxygenase products.…”

Transition Metal Complexes and the Activation of Dioxygen

“…Related pathways are implicated for non-heme iron enzymes that are supported by three histidine ligands instead of the 2-His-1-carboxylate triad [174], such as cysteine dioxygenase [175][176][177] and β-diketone dioxygenase [178]. Also notable is the chemistry of a low-spin Fe(III)-OOH species identified in the anticancer drug bleomycin, which may perform hydrogen atom abstraction from deoxyribonucleic acid directly or via prior O-O bond scission pathways [179,180].…”